Olfactory Stem Cells Reveal MOCOS As a New Player in Autism Spectrum Disorders
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OPEN Molecular Psychiatry (2016) 21, 1215–1224 www.nature.com/mp ORIGINAL ARTICLE Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders F Féron1,2,10, B Gepner2,10, E Lacassagne2, D Stephan2, B Mesnage2, M-P Blanchard3, N Boulanger4, C Tardif5, A Devèze6,7, S Rousseau8, K Suzuki9, JC Izpisua Belmonte9, M Khrestchatisky2, E Nivet2 and M Erard-Garcia2 With an onset under the age of 3 years, autism spectrum disorders (ASDs) are now understood as diseases arising from pre- and/or early postnatal brain developmental anomalies and/or early brain insults. To unveil the molecular mechanisms taking place during the misshaping of the developing brain, we chose to study cells that are representative of the very early stages of ontogenesis, namely stem cells. Here we report on MOlybdenum COfactor Sulfurase (MOCOS), an enzyme involved in purine metabolism, as a newly identified player in ASD. We found in adult nasal olfactory stem cells of 11 adults with ASD that MOCOS is downregulated in most of them when compared with 11 age- and gender-matched control adults without any neuropsychiatric disorders. Genetic approaches using in vivo and in vitro engineered models converge to indicate that altered expression of MOCOS results in neurotransmission and synaptic defects. Furthermore, we found that MOCOS misexpression induces increased oxidative-stress sensitivity. Our results demonstrate that altered MOCOS expression is likely to have an impact on neurodevelopment and neurotransmission, and may explain comorbid conditions, including gastrointestinal disorders. We anticipate our discovery to be a fresh starting point for the study on the roles of MOCOS in brain development and its functional implications in ASD clinical symptoms. Moreover, our study suggests the possible development of new diagnostic tests based on MOCOS expression, and paves the way for drug screening targeting MOCOS and/or the purine metabolism to ultimately develop novel treatments in ASD. Molecular Psychiatry (2016) 21, 1215–1224; doi:10.1038/mp.2015.106; published online 4 August 2015 INTRODUCTION Our study is based on a relatively homogeneous cohort of nine Autism spectrum disorders (ASDs) are complex neurodevelop- adults with severe autism and low to very low developmental mental diseases arising from multifactorial genetic, epigenetic and disabilities plus two adults with mild or moderate autism and no environmental origins.1–4 Systems-level connectivity features5 and or mild cognitive abilities (Asperger syndrome or high-functioning plausible neuroanatomical,6 cellular7,8 and molecular9 underpin- autism), paired with 11 age- and gender-matched control nings of ASD have been highlighted. Although hundreds of individuals. We first chose a non-hypothesis-driven approach susceptibility genes have been identified, being generally involved and looked for transcriptome anomalies, using pangenomic cDNA in neurobiological functions such as neurogenesis, synaptogenesis microarrays. We observed a dysregulated expression of genes and neurotransmission,10 collectively they account for 10–20% of already associated to ASD. Of note, for the very first time, we also ASD cases at most.11 Accordingly, discovering common genetic identified a new candidate gene, the MOlybdenum COfactor traits being more representative of ASD population becomes a Sulfurase (MOCOS). MOCOS is known as an enzyme of the purine priority for early diagnoses,12 investigation of ASD physiopathology metabolism that sulfurates the molybdenum cofactor, thus and identification of new therapeutic targets. allowing the two downstream enzymes—xanthine dehydrogen- ASD-associated profiles are underpinned by atypical neural ase (XDH) and aldehyde oxidase (AOX1)—to be active.17 development often accompanied by epilepsy, gastrointestinal Considering the role of XDH and AOX in purine degradation as disorders and other comorbid disorders.3 Despite numerous well as in the Redox balance, and so in cellular stress response, the studies, the etiopathology as well as the physiopathogeny of these role of MOCOS appears to be important though poorly disorders remain largely elusive. To date, genetic studies on ASD investigated yet. MOCOS mutations have been associated with have mainly used cellular material that, even though being xanthinuria type II, a disorder characterized by an abundant sufficient for mutagenic studies, might be irrelevant to identify excretion of urinary xanthine and a diminished production of uric – gene misexpression during development. To seek for novel acid, in both serum and urine.18 20 To date, however, nothing is candidates having a role in ASD, we chose to study cells that are known about a putative role of this gene on cerebral functions. representative of early stages of ontogenesis. To this end, we Therefore, and considering the clinical symptoms associated to decided to use human nasal olfactory stem cells (OSCs) displaying ASD, our data led us to study whether a dysregulation of MOCOS multipotent properties13,14 andprovedtobeusefulfortranscrip- expression could have an impact on functions known to be tomic analyses in the context of brain disorders.15,16 involved in the related disease phenotype. 1Inserm CBT 1409, Centre d'Investigations Cliniques en Biothérapie, Marseille, France; 2Aix Marseille Université, CNRS, NICN UMR 7259, Marseille, France; 3Aix Marseille Université, CNRS, CRN2M UMR 6231, Marseille, France; 4Aix Marseille Université, TAGC UMR 1090, Marseille, France; 5Aix Marseille Université, PsyCLE, EA 3273, Aix en Provence, France; 6AP-HM, Département ORL, Marseille, France; 7Aix Marseille Université IFSTTAR, UMRT 24, Marseille, France; 8AP-HM, Département Anesthésie, Marseille, France and 9Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA. Correspondence: Professor F Feron or Dr M Erard-Garcia, Faculty of Medicine, Aix Marseille Université, CNRS, NICN UMR 7259, 13344 Marseille, France. E-mail: [email protected] or [email protected] 10These authors contributed equally to this work. Received 28 January 2015; revised 6 May 2015; accepted 15 June 2015; published online 4 August 2015 MOCOS, a new candidate in ASD F Féron et al 1216 We found that the genetic ablation of mocs-1 (the MOCOS frame deletion of the L-cysteine desulfurase domain: ok3439 is predicted to ortholog) in Caenorhabditis elegans (C. elegans) induces an be devoid of 163 amino acids in a region known to bind the PLP cofactor alteration of the response to oxidative stress and is responsible and mobilize sulfur from L-cysteine. Thus, ok3439 deletion mutants are for abnormal neurotransmission phenotypes. Then, we confirmed supposed to be free of their cysteine desulfurase activity, whereas the MOSC these data in human cells. First, in OSCs that displayed a higher domain remains intact. The ok1816 mutant is a deletion mutant for F15E6.6. C57/Bl6 mice (8-week old) were used to evaluate the expression of production of reactive oxygen species (ROS). Second, in human MOCOS in various organs as well as to establish primary cultures of neurons. induced pluripotent cells (iPSCs) in which the gene MOCOS was All murine experiments were conducted according to the guidelines of the partially knocked down and showed a reduced synaptogenesis Ethics Committee of the Medical Faculty of Marseille and conform to when differentiated into neurons. National and European regulations (EU directive Nu 86/609). Animals were housed in air-flow racks on a restricted access area and maintained on a 12- h light/dark cycle at a constant temperature (22 ± 1 °C). For animal studies, MATERIALS AND METHODS no method of randomization was used and no blinding was done. Participants A complete written and oral information on the goal and procedure of this Cell culture research was provided to the participants or their legal tutors and a signed All cell culture-related works were performed as described in informed consent was obtained from all of them, before their involvement Supplementary Information Methods. in the study. All procedures were approved by the local ethical committee (Comité de Protection des Personnes, files #205016 and #205017) of Marseille. Patients Microarray gene expression analysis with ASD (n = 11) were recruited in three public hospital units OSCs from 22 individuals were collected and processed for hybridization (Valvert Hospital, Marseille; Montperrin Hospital, Aix-en-Provence and on pangenomic cDNA arrays and microarray data analysis as described in Edouard Toulouse Hospital, Marseille), in two socio-medical private units Supplementary Information Methods. Microarray data have been depos- (Foyer d’accueil médicalisé, Pélissanne and Service d’accompagnement ited in NCBI-GEO under the accession number GSE63524. medico-social pour adultes autistes, Salon de Provence) and through a specialized ASD diagnosis consultation (Montperrin Hospital, Aix-en- RNA isolation and real-time PCR analysis Provence). The patients were diagnosed according to ICD-10 (World Health Organization, 1993) and DSM-5 (American Psychiatric Association, RNA samples extracted from OSCs were tested with qRT-PCR as described 2013) criteria for pervasive developmental disorders and ASDs, respec- in Supplementary Information Methods. tively. Control patients were recruited to match the age and gender of each ASD patient that was endorsed in the cohort. The healthy controls Western blot analysis were neither presenting a neuropsychiatric disorder nor taking medication. Protein expression analyses in OSCs